SPECTROPHOTOMETRY

DEFINITION
Spectrophotometry = an analytical method that uses
electromagnetic radiation for measurement of
chemical concentrations.

The principle of spectrophotometry is based on the

physical laws of radiant energy or light.
Types of spectrophotometry

1. Measurement of absorbed or
transmitted light.

2. Measurement of emitted light.

Fundamentals of Spectrophotometry
1.) Colorimetry
 An analytical technique in which the concentration of
an analyte is measured by its ability to produce or
change the color of a solution
- Changes the solution’s ability to absorb light

2.) Spectrophotometry
 Any technique that uses light to measure chemical
concentrations
 A colorimetric method where an instrument is used to
determine the amount of analyte in a sample by the
sample’s ability or inability to absorb light at a certain
wavelength.
 Electromagnetic Radiation:
Properties of light and radiant energy

Electromagnetic radiation is described as

photons of energy traveling in waves

There is a relationship between energy and the

length of the wave (wavelength)

The more energy contained, the more frequent

the wave and therefore, the shorter the
wavelength

5
 Electromagnetic Radiation:
Properties of light and radiant energy
 This relationship between energy and frequency is expressed
by Planck's formula:

 E = hf
Where: E= energy of a photon
h = a constant
f = frequency

 The formula shows that the higher the frequency; the higher the
energy or the lower the frequency, the lower the energy

 We do not use this to perform any calculations. You only need to

recognize Planck’s formula and its components 6
Properties of Light
Particles and Waves

 Light waves consist of perpendicular, oscillating electric and magnetic fields

 Parameters used to describe light

- Amplitude (A): height of wave’s electric vector

- Wavelength (l): distance (nm, cm, m) from peak to peak

- Frequency (n): number of complete oscillations that the waves makes

each second
 Hertz (Hz): unit of frequency, second-1 (s-1)
 1 megahertz (MHz) = 106s-1 = 106Hz
The electromagnetic spectrum
• The ultraviolet region extends from about 10 to 380 nm
•The near - ultraviolet region or quartz UV region is from
200 to 380 nm
• The visible (VIS) region extends from about 380 to 780 nm
•The infrared (IR) region extends from about 0.78μm to 300
μm
•The near-infrared (IR) region extends from about 0.80μm
to 2.5 μm
•The far-infrared (IR) region extends from about 2.5μm to
16 μm
Electromagnetic Spectra

9
 The absorption process
How does matter absorb radiation

When polychromatic light (white light), which contains the whole

spectrum of wavelengths in visible region, is passed though an object
will absorb certain of the wavelengths, leaving the unabsorbed
wavelengths to be transmitted. These residual transmitted wavelengths
will be seen as a color.

This color is complementary to the absorbed colors.

Absorption of Light
Colors of Visible Light
 Many Types of Chemicals Absorb Various Forms of Light

 The Color of Light Absorbed and Observed passing through the

Compound are Complimentary
Visible region wavelength

Color Wavelength (nm)

Ultraviolet 400 and under
Violet 400 - 450
Blue 450 - 500
Green 500 - 570
Yellow 570 - 590
Orange 590 - 620
Red 620 - 650
Infrared 750 & over
Absorption of Light

Ground and Excited State

 When a chemical absorbs light, it goes from a low energy state
(ground state) to a higher energy state (excited state)

Energy required of photon

to give this transition:
DE = E1 - Eo

 Only photons with energies exactly equal to the energy difference

between the two electron states will be absorbed

 Since different chemicals have different electron shells which are

filled, they will each absorb their own particular type of light
- Different electron ground states and excited states
Beer-Lambert’s Law

The fraction of the incident light absorbed by a

solution at a given wavelength is related to:

a. thickness of the absorbing layer

(path length)

and

b. concentration of the absorbing species

• Beer-Lambert’s Law

I 
log   = cl
I 
Where:
 o

Io = intensity of incident light

I = intensity of transmitted light
 = molar extinction coefficient
c = concentration of the absorbing species (mol/L)
l = path length of the light-absorbing sample (cm)
Transmittance
• Defined as the ratio of the intensity of light
emerging from the solution (I) to that of
incident light entering (Io)

T= I
Io Io I
Absorption of Light

 The relative amount of light making it through the

sample (I/Io) is known as the transmittance (T)

I
T=
Io

I 
Percent transmittance %T = 100   
 Io 
T has a range of 0 to 1, %T has a range of 0 to 100%
Transmittance

100% transmittance means no light is

absorbed by the solution so that incident light
is 100% transmitted
Absorption of Light

 Absorbance (A) is the relative amount of light absorbed by

the sample and is related to transmittance (T)
- Absorbance is sometimes called optical density (OD)

I 
A = - log  = - log(T ) = - log(%T / 100)
 Io 

A has a range of 0 to infinity

Spectrophotometer
Basic Design
 An instrument used to make absorbance or
transmittance measurements is known as a
spectrophotometer
Spectrophotometer: Components
Light source/lamps
Vary according to need, but must be a constant beam, cool and
orderly
Types
Tungsten or tungsten iodide lamps for visible and near
infrared
Incandescent light (400 nm - 700 nm)
Deuterium or mercury-arc lamps required for work in U.V.
range
Range 160-375 nm
Spectrophotometer: Components
Monochromators
Promote spectral isolation
Operator selects specific wavelength
Isolate a single wavelength of light
Provides increased sensitivity & specificity

Types
Glass filters
Prisms
Diffraction gratings
Spectrophotometer: Components
Cuvette
– Made of high quality glass or quartz
• Glass – for work in the visible light range
• Quartz or fused silica – for work in the UV
range
– Shape
• Round cuvettes are cheaper but light
refraction and distortion occur
• Square cuvettes have less light refraction but
usually more costly
– Optically clean
• No inconsistencies in composition
• No marks, scratches, or fingerprints
– Positioning
• Orientation and placement into the instrument
important. Each time must be the same so
light passes through the cuvet at the same
place.
Spectrophotometer: Components
Photodetectors

Purpose – to convert the transmitted light

into an equivalent amount of electrical
energy
Most common is the photomultiplier tube
Spectrophotometer: Components
Readout devices

Purpose – to convert the electrical

signal from the detector to a
usable form

Types
Meters/Galvanometers
Recorders
Digital Readout
1. Turn instrument on

2. Select correct wavelength 

3. Block light, set Zero (no light = infinity

absorption = 0% T)

4. Choose and clean cuvette

5. Open light, insert Blank (maximum light = no

absorption = 100% T)

6. Measure absorption of Standards, Controls and

Patient samples to 3rd decimal place
Standards
• Precisely prepared = known concentration

• Usually pure solution of single compound

• Plot absorbance vs concentration: standard

curve
 Calibration Curve
• Definition: calibration curve for a certain
compound represents the graphical representation of
the absorbance as a function of concentration

• How to prepare a calibration curve – by reading the

absorbances of 3-10 standard solutions
Blank Solution
A mixture of all reagents, except the compund X of
interest

It is use to set the photometer to 0 absorbance (A) or

100% transmittance (%T)

ROLE: to account for imperfections in the cuvette

and to cancel readings due to the absorbance of the
reagents
Sample
Solution containing the compound X
of interest with an unknown
concentration
 Calculation

Cu = Cs x A(u) x D
A(s)
Where: Cs = concentration of standard
Cu = concentration of unknown
A(s) = absorbance of standard
A(u) = absorbance of unknown
D = dilution factor
 Calibration Curve

Glucose Standard Calibration Curve

Glucose Absorba
Std. Concn. nce 1.2
60 mg% 0.2 1

Absorbance
0.8
120 mg% 0.4 0.6
0.4
0.2 ) ( Linear
U 0.5
0
60 120 180 200
180 mg% 0.6
Mg% glucose
Hemoglobin Concentration
Determination
 Hemoglobin (Hb)
• Hemoglobin (Hb) is the standard abbreviation for
hemoglobin, the oxygen-carrying pigment and
predominant protein in the red blood cells.

• Hemoglobin is the protein that carries oxygen

from the lungs to the tissues and carries carbon
dioxide from the tissues back to the lungs.
 Structure of hemoglobin
• A hemoglobin molecule consists of
four polypeptide chains: two alpha
chains, each with 141 amino acids
and two beta chains, each with 146
amino acids. The protein portion of
each of these chains is called
"globin".

• The α and β globin chains are very

similar in structure and each one of
them is liked with a heme molecule.
 Heme molecules
• A heme group is a flat ring
molecule containing carbon,
nitrogen and hydrogen atoms,
with a single Fe2+ ion at the
center. Without the iron, the ring
is called a porphyrin.
 Normal Ranges
• In the very common laboratory test for hemoglobin (Hb), it is
measured as total hemoglobin and the result is expressed as the
amount of hemoglobin in grams (gm) per deciliter (dl) of whole
blood, a deciliter being 100 milliliters.

• The normal ranges for hemoglobin depend on:

• The age.
• Altitude
• The sex of the person.
 The normal ranges
Newborns 17-22gmdl
One (1) week of age 17-22gm\dl
One (1) week of age 15-20gmdl
One (1) month of age 11-15gm\dl
Children 11-13gm\dl
Adult men 14-18gm\dl
Adult women 12-16gm\dl
Men after middle age 12.4-14.9gm\dl
Women after middle age 11.7-13.8gm\dl
• Normal values in an adult are 12 to 18 grams per deciliter
(100 milliliters) of blood.
• Above-normal hemoglobin levels is called polycythemia
which is may be:

• Secondary polycythemia which is may be due to:

– Dehydration (sever burns, diarrhea, vomitting, …etc.).
– Severe lung or heart disease.
– Living at high altitudes.
– Heavy smoking.

• Primary polycythemia which is due malignant variation

in blood cells production in bone marrow
• Below-normal hemoglobin levels may lead to anemia that can be
the result of:

– Iron deficiency or deficiencies in essential vitamins of other

elements, such as B12, folate, B6.
– Inherited hemoglobin defects, such as sickle cell anemia or
Thalassaemia.
– Other inherited defects affecting the red blood cells.
– Excessive bleeding.
– Excessive destruction of red blood cells.
– Kidney disease.
– Bone marrow failure or aplastic anemia.
– Cancers that affect the bone marrow.
 Measurement of hemoglobin

• The Cyanmethemoglobin Method for Hb determination is the

reference method.

• Principle:
• Whole blood is diluted in a solution of potassium Ferricyanide
and potassium cyanide.
• The Hb is oxidize to methemoglobin by the Ferricyanide.
• The potassium cyanide then converts the methemoglobin to
cyanmethemoglobin.
• The absorbance of the cyanmethemoglobin at 540 nm is
directly proportional to the Hb concentration.
• Sulfhemoglobin is not converted to cyanmethemoglobin;
therefore, it can not be measured by this method.
• Hb (Fe++) K3Fe (CN)6 methemoglobin (Fe+++ )

KCN Cyanmethemoglobin